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Detection of high Leishmania infantum loads in Phlebotomus perniciosus captured in the leishmaniasis focus of southwestern Madrid region (Spain) by real time PCR
Accepted Manuscript Title: Detection of high Leishmania infantum loads in Phlebotomus perniciosus captured in the leishmaniasis focus of southwestern Madrid region (Spain) by real time PCR ´ Authors: Estela Gonz´alez, Ana Alvarez, Sonia Ruiz, Ricardo Molina, Maribel Jim´enez PII: DOI: Reference:
S0001-706X(16)31049-X http://dx.doi.org/doi:10.1016/j.actatropica.2017.03.023 ACTROP 4249
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
2-12-2016 20-3-2017 24-3-2017
´ Please cite this article as: Gonz´alez, Estela, Alvarez, Ana, Ruiz, Sonia, Molina, Ricardo, Jim´enez, Maribel, Detection of high Leishmania infantum loads in Phlebotomus perniciosus captured in the leishmaniasis focus of southwestern Madrid region (Spain) by real time PCR.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.03.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Detection of high Leishmania infantum loads in Phlebotomus perniciosus captured in the leishmaniasis focus of southwestern Madrid region (Spain) by real time PCR Estela González, Ana Álvarez, Sonia Ruiz, Ricardo Molina, Maribel Jiménez§ Unidad de Entomología Médica, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo s/n, 28220 Majadahonda, Madrid, Spain §
Corresponding author. Tel.: +34 91 822 3674; fax: +34 91 509 7034.
E-mail address: [email protected] (Maribel Jiménez)
Graphical abstarct In this work a qPCR assay was applied in order to estimate the number of parasites in P. perniciosus females collected in the human leishmaniasis focus that is affecting southwestern Madrid region
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Highlights
A real time PCR was applied in female sand flies captured in a human leishmaniasis focus
Limit of detection achieved was 0.001 parasite/reaction, corresponding to 0.08fg of parasite DNA
High parasite loads (>1000 parasites/reaction) in sand flies from the focus were observed
Unfed females showed significant higher parasite loads than blood-fed females
Abstract Since 2010 a human leishmaniasis outbreak has been notified in southwestern Madrid region that still remains active. Entomological surveys have been carried out in the affected area in order to obtain information about species diversity, distribution, and density of sand flies. Moreover, molecular identification of blood meal preferences of sand flies and molecular detection of Leishmania infantum has been performed. In this work, we optimized a real time PCR assay in order to determine parasite loads in unfed and blood-fed Phlebotomus perniciosus female sand flies caught in the focus area. Results showed elevated parasite loads in nearly 70% of the studied positive sand flies. Furthermore, significantly higher parasite loads were observed in females without blood in their guts. In conclusion, high L. infantum loads found in P. perniciosus sand flies from the Madrid focus support the exceptional characteristics of this outbreak. 2
Keywords: Phlebotomus perniciosus, qPCR, Leishmania infantum loads, human leishmaniasis outbreak of Madrid
1. Introduction Leishmaniasis is a globally spread vector-borne disease caused by trypanosomatids of genus Leishmania and transmitted by phlebotomine sand fly bites. Southern Europe region is considered as a hypoendemic region for leishmaniasis (World Health Organization, 2010). In particular, Spain has registered an increase of visceral and cutaneous leishmaniasis human cases since 2010 when an outbreak of this disease located in the southwest of the Community of Madrid was detected (Arce et al., 2013; Gomez-Barroso et al., 2015). Since then, several studies have been performed in order to characterize this outbreak. Different factors related to the urbanization and changes in land use in the affected area of Madrid have been involved in the hypothesis about this leishmaniasis re-emergence, favoring the parasite transmission among wild animals of the green areas surrounding the municipalities affected by the outbreak. In this way, studies carried out elsewhere suggest that human intervention in many areas leads to a change in the transmission cycle of parasites by increasing exposure of infected vectors and susceptible reservoirs (Harhay et al., 2011). Hares and rabbits from the aforementioned region have been incriminated in the transmission of Leishmania infantum, demonstrating that a sylvatic cycle has been taking place (Jiménez et al., 2014; Molina et al., 2012). On the other hand, Leishmania parasites isolated from patients from the 3
focus, as well as from wild reservoirs and sand flies collected in the same area, have been characterized by molecular procedures showing the same genotype of L. infantum (Chicharro et al., 2013; Jiménez et al., 2014; Molina et al., 2012). Additionally, a great virulence of the L. infantum strain circulating in the focus area has been reported (Domínguez-Bernal et al., 2014). Systematic entomological captures have been carried out in the focus since 2011 demonstrating that the predominant sand fly species is Phlebotomus perniciosus (Alten et al., 2016; Tello et al., 2015), which is the main vector of leishmaniasis in Spain. Both molecular detections by conventional PCR and dissections performed with P. perniciosus females collected throughout these surveys show high L. infantum infection rates, although parasite loads have not been determined (Jiménez et al., 2014, 2013; data not published). At present, many studies have been performed for diagnostic purposes by using real time PCR (qPCR) assay for Leishmania detection and quantification. Thus, a qPCR targeting kDNA detection in fresh blood and tissue has also been developed by Mary et al., (2004) and Nicolas et al., (2002). Similarly, Abbasi et al., (2013) developed a qPCR in order to detect and quantify Leishmania donovani in human dried-blood. Leishmania species discrimination has been studied by qPCR in human samples (Weirather et al., 2011). Moreover, parasite loads in spleen have been correlated with blood parasite burden in visceral leishmaniasis using qPCR (Sudarshan et al., 2015). Even more, some authors searching for non-invasive methods have developed qPCR assays in order to detect and quantify Leishmania parasites in hair and earwax of dogs (Belinchón-Lorenzo et al., 2016, 2013). On the other hand, there are several works focused on the study of parasite burden in sand fly vectors. Myskova et
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al., (2008) describe for the first time a qPCR method to determine the parasite loads in Phlebotomus duboscqi. Since then, qPCR has been applied in other sand fly species such as Phlebotomus sergenti of Iran (Aghaei et al., 2014); Phlebotomus papatasi and Phlebotomus alexandri of Iraq (Coleman et al., 2009), Phlebotomus perfiliewi, P. perniciosus, and Phlebotomus neglectus of Italy (Dantas-Torres et al., 2014), and Lutzomyia longipalpis of Brazil (BezerraVasconcelos et al., 2011; Cunha et al., 2014). Undoubtedly, knowledge of parasite loads in sand flies may provide important information about transmission patterns in a particular region. Furthermore, it is well known that Leishmania parasites can manipulate sand fly feeding behavior. Thus, high parasite loads in sand fly midguts are correlated to a persistent feeding pattern that leads to an increase in Leishmania transmission (Rogers and Bates, 2007). In the present work, we applied and validated a qPCR assay to detect and estimate the number of L. infantum parasites in the gut of P. perniciosus females caught in the aforementioned human leishmaniasis focus that is affecting southwestern Madrid region.
2. Materials and Methods 2.1.
Leishmania parasites
The L. infantum strain IPER/ES/2012/POL2FL7, isolated from a naturally infected sand fly captured in the leishmaniasis focus of southwestern Madrid region (Spain) and characterized as ITS-Lombardi genotype, was used in this study. The promastigotes were cultured in RPMI medium supplemented with 20 % inactivated fetal calf serum (FCS, HycloneTM, GE Healthcare Life Science, NJ, USA) and a mixture of penicillin-streptomycin (10000 U/ml, Lonza 5
BioWitthaker®, Verviers, Belgium). L. infantum promastigotes were collected by centrifugation and resuspended in buffered saline. Then, promastigotes suspension was quantified by duplicate using a Neubauer® chamber in order to obtain 107 promastigotes/ml. Subsequent 10-fold dilutions were carried out. 2.2.
Samples
DNA samples obtained from 135 female sand flies collected in the entomological surveys carried out from June to October 2012, 2013, and 2014 were analyzed by qPCR. These samples were previously examined by conventional kDNA-PCR finding 68 positive sand flies for Leishmania DNA. Sixty-four out of the 135 analyzed sand flies showed indication of a recent blood meal. Sand flies reared in the Medical Entomology Unit of the Carlos III Health Institute, Spain, were used as negative control. 2.3.
DNA isolation
DNA from promastigotes dilutions were extracted using Blood and Tissue DNA extraction Kit (Qiagen®) following manufacturer instructions. On the other hand, DNA from reared and field-captured sand flies was obtained using the abovementioned kit and following the protocol previously described (Jiménez et al., 2013). 2.4.
Determination of parasite loads by qPCR
Kinetoplast minicircle primers JW11: 5’-CCT ATT TTA CAC CAA CCC CCA GT-3’ and JW12: 5’-GGG TAG GGG CGT TCT GCG AAA-3’ (Nicolas et al., 2002) were used for Leishmania detection in both conventional and qPCR methods. Different assays were performed in order to enhance primer
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concentration and the number of cycles of qPCR. Finally, each reaction consisted in a final volume of 20 µl, containing 10 µl of Quantimix Easy Kit (Biotools®), 0.1 µM of each primer, 0.5 µl of BSA (Roche®), and 30 ng of DNA extracted from each sand fly. PCR amplification was performed as follows: an initial incubation at 95ºC for 15 min followed by 40 cycles at 95ºC for 10 s, 58ºC for 10 s and a final extension at 72ºC for 30 s. At the end of each cycle, SYBR® Green I fluorescence acquisition was performed. Non template control (NTC), negative control (NC, reared sand flies) and positive controls (two promastigotes dilutions mixed with 30 ng of sand fly DNA) were added in each reaction. Amplifications were carried out in a Corbett Rotor-GeneTM 6000 realtime PCR System (Qiagen®). 2.5.
Data analysis and statistics
Threshold cycle (Ct) values were calculated by Rotor-Gene Series Software version 1.7 using default parameters. Standard curves using DNA obtained from promastigotes dilutions and DNA from promastigotes dilutions mixed with 30 ng of DNA from reared sand flies were performed by triplicate. Mean and standard deviation (SD) of Ct values of each standard curve dilution were calculated. Non parametric statistical Kruskal-Wallis method was used to calculate significant differences between Ct values in the three replicates of each dilution of the standard curves. The same statistical test was applied to study differences in parasite loads between the three periods. Meanwhile, MannWhitney method was used to study differences between Ct values of both standard curves. This statistical method was also applied to analyze parasite loads differences among blood-fed and unfed female sand flies. Differences 7
were considered statistically significant when p-values were less than 0.05, using GraphPad Prism 6.07 software.
3. Results 3.1.
Sensitivity and Efficiency
With the aim to optimize this qPCR, several experiments were carried out using different primer concentration and number of cycles per reaction. In this assay, we observed that a number of cycles greater than 40 produced a visible smear in the agarose gel and a lower efficiency. On the other hand, a primer concentration higher than 0.1 µM resulted in dimers presence. To analyze the sensitivity of the reaction, amplification of 10-fold dilutions ranged from 107 to 1 promastigote/ml was carried out by triplicate. The limit of detection (LOD) was 0.001 parasite/reaction, which corresponds to 1 promastigote/ml from the original sample (1 µl was used per reaction). A second standard curve mixing DNA extracted from each promastigote dilution and 30 ng of DNA from reared sand flies was performed. The LOD obtained in both standard curves was the same. Based on the haploid genome size of L. infantum (32.1Mb, 69.7 fg) and adding ≃10.5 fg of kinetoplast genomic material, one parasite yield ≃80.2 fg (Peacock et al., 2007). According to this, our qPCR assay achieved a LOD of 0.08 fg of parasite DNA. On the other side, reproducibility in both standard curves was assessed and replicates of each dilution did not show significant differences according to statistical results (pvalues = 0.8981 and 0.8847 for DNA promastigote dilutions and DNA from promastigotes mixed with sand fly DNA, respectively). In the same way, there 8
was no significant difference between both standard curves (p-value = 0.6294), showing no inhibition of DNA from reared sand flies in the reaction (Table 1). Finally, standard curves showed loss of linearity and efficiency when dilutions below 1 parasite/reaction were used (Fig. 1). The efficiency showed optimum values (average slope= -3.2±0.14; average efficiency= 1.06±0.07) in case of qPCR reactions of samples (Fig. 2). 3.2.
Leishmania quantification in field-captured sand flies
A total of 135 DNA samples of female sand flies captured in the outbreak area in southwestern Madrid were analyzed by the qPCR optimized in this study. Results by qPCR did not present differences with conventional PCR, detecting n= 68 positive samples (35 blood-fed and 33 unfed) and n= 67 negative samples (29 blood-fed and 38 unfed females) by both methods. Ct threshold values were calculated according to default parameters and NTC Ct values in each qPCR reaction were employed as the cut-off value for discrimination between positive and negative samples (Fig. 2). The 68 positive samples were classified in different categories according to the parasite loads: 16.17% (n=11) showed very high loads (>10,000 parasites/reaction; a specimen showing over than 100,000 parasites/reaction is included in this category), 23.53% (n = 16) showed high loads (>1,000 parasites/reaction), 30.9% (n = 21) presented moderate loads (10-1000 parasites/reaction) and 29.4% (n = 20), low parasite loads (<10 parasites/reaction) (Table 2). Parasite burden were higher in sand flies caught in 2012, although differences were not significant (p-value = 0.7233).
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Mean parasite load in blood-fed females was 8.02 parasites/reaction, while in unfed females was 1,580 parasites/reaction. In this case, the differences are significant (p-value < 0.0001).
4. Discussion This study presents the validation of a qPCR assay for the detection of L. infantum and the quantification of parasite loads using sand flies collected in a periurban area of southwestern Madrid affected by a leishmaniasis outbreak since 2010 (Arce et al., 2013; Gomez-Barroso et al., 2015). A high abundance of P. perniciosus sand fly, the most important vector of leishmaniasis in western Mediterranean basin, has been found in this focus (Aránguez et al., 2014; Arce et al., 2013; Tello et al., 2015; Alten et al., 2016). Additionally, other studies revealed some special features of this focus like the existence of a sylvatic cycle of L. infantum involving lagomorphs living in the green areas surrounded by the aforementioned urban area (Jiménez et al., 2013, 2014; Martín-Martín et al., 2014; Molina et al., 2012) and the high virulence of the L. infantum isolates obtained from sand flies captured in the same areas (Domínguez-Bernal et al., 2014; Martín-Martín et al., 2015). Even though elevated Leishmania infection rates have been observed by dissection (data not published) and conventional PCR (Jiménez et al., 2013) among P. perniciosus females collected in the focus, no information was available on their parasite loads to date. In the present work, the qPCR showed the same sensitivity as the conventional PCR performed on the same samples. The LOD has been established in 0.001 parasites/reaction with the qPCR developed, corresponding to 0.08 fg of parasite DNA. Similar LOD values have been reported by others using qPCR in 10
sand flies targeting kDNA (Bezerra-Vasconcelos et al., 2011; Cunha et al., 2014; Myskova et al., 2008). In spite of the good LOD, standard curves showed less linearity in dilutions lower than 1 parasite/µl, as has been reported by Abbasi et al., (2013) when assessing the efficacy of qPCR for detecting L. donovani in human dried-blood samples. Nevertheless, reactions performed in the present work with sand fly samples demonstrated good efficiency and parasite burden was successfully estimated. Seventy percent of the sand flies analyzed in this study presented a significant number of Leishmania parasites in their guts and 16.17% of them showed large infections greater than 10,000 parasites/reaction, and what is more, a specimen presented more than 100,000 parasites/reaction. Such parasite loads found in wild caught P. perniciosus females had not been reported so far. Only studies carried out in wild L. longipalpis have reported higher parasite loads (Rodrigues et al., 2016). The exceptional features observed in the outbreak area as the high density of lagomorphs (Jiménez et al., 2014), the high L. infantum infection rates in spleen and skin samples of hares (29.9%) and rabbits (21.2%) (Community of Madrid data), and the elevated proportion of sand flies engorged with blood of rabbits or hares in the green park (Jiménez et al., 2013, 2014; González et al., 2015), may have given rise to the high parasite loads detected in wild caught sand flies, thereby increasing the transmission rates of the parasite. In addition, a large number of promastigotes can block more easily the stomodeal valve of sand flies forcing them to remain feeding for longer on the same or different hosts to often obtain only a partial meal of blood which increases the likelihood of parasite transmission. In this sense, experimental infections have proven that both L. infantum and L. mexicana promote feeding 11
on multiple hosts (Rogers and Bates, 2007). Moreover, the infectious dose may be one of the determining factors in the outcome of Leishmania infection (Kimblin et al., 2008; Maia et al., 2011). The significantly higher parasite loads found in females without blood in their guts are undeniable proof of a proper establishment and replication of the parasites that would be more likely to survive and develop late-stage infections in the sand fly midgut (Roger and Bates, 2007, Myskova et al., 2008). The intensity of infection in wild caught sand flies gives evidence of parasite growth and possible transmission (Killick-Kendrick, 1990). In other words, P. perniciosus is an excellent vector of leishmaniasis in the outbreak area. Similarly, other authors have found in India higher parasite loads in gravid and unfed sand flies than in blood-fed sand flies (Tiwary et al., 2013). As for the low parasite loads found in most of the blood-engorged sand flies studied could be due to a recent ingestion of blood on infecting vertebrates from which these uninfected flies had fed. Finally, the high parasite loads observed in some females with freshly ingested blood in their guts suggest that they could be already infected at the time of the next blood ingestion and potentially transmit the parasite to the vertebrate from which they had fed. These findings could give us an idea about the intensity of transmission of the parasite from wild vertebrates to sand flies in the area. Although the quantification of parasite burden in sand flies by qPCR can be suitable for the incrimination of suspected vectors of leishmaniasis, it would be advisable to combine with sand fly dissection in order to determine the localization of the promastigotes in the gut and to isolate the leishmania strain (Myskova et al., 2008).
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In conclusion, the qPCR assay validated in the present study has allowed the detection and quantification of elevated parasite loads in a high number of the P. perniciosus female sand flies captured in the human leishmaniasis focus that is affecting southwestern Madrid region. These results are further evidence of the exceptional features of this leishmaniasis focus which require special attention by establishing an intensive and ongoing surveillance program of the disease in the outbreak area.
Conflict of interest The authors declare no conflicts of interest.
Acknowledgments This study was partially funded by the Dirección General de Salud Pública, Consejería de Sanidad of Community of Madrid, Spain and the Instituto de Salud Carlos III, Madrid, Spain. We want to thank Sonia Hernández for technical assistance.
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Bringaud, F., Baldauf, S.L., Faulconbridge, A., Jeffares, D., Depledge, D.P., Oyola, S.O., Hilley, J.D., Brito, L.O., Tosi, L.R.O., Barrell, B., Cruz, A.K., Mottram, J.C., Smith, D.F., Berriman, M., 2007. Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat. Genet. 39, 839–47. doi:10.1038/ng2053 Rodrigues, A.C.M., Magalhães, L. M., Damasceno, R.F., Batista de Moraes, N., Souza Junior, A.D., Bevilaqua, C.M.L., 2016. Molecular identification of Lutzomyia migonei ( Diptera: Psychodidae ) as a potential vector for Leishmania infantum ( Kinetoplastida: Trypanosomatidae ). Vet. Parasitol. 220, 28-32. doi: 10.1016/j.vetpar.2016.02.018 Rogers, M.E., Bates, P. 2007. Leishmania manipulation of sand fly feeding behavior results in enhanced transmission. PLoS Pathog. 3, 818–825. doi:10.1371/journal.ppat.0030091 Sudarshan, M., Singh, T., Chakravarty, J., Sundar, S., 2015. A correlative study of splenic parasite score and peripheral blood parasite load estimation by qPCR in visceral leishmaniasis. J. Clin. Microbiol. JCM.01465–15. doi:10.1128/JCM.01465-15 Tello, A., González-Mora, D., Outerelo, R., Iriso, A., Ángeles, M., 2015. Los flebotomos del brote de leishmaniasis en el suroeste de la Comunidad de Madrid (Diptera, Psychodidae, Phlebotominae) The sand flies of the outbreak of leishmaniasis in south-west area of Madrid. Bol. R. Soc. Esp. Hist. Nat. Sec. Biol., 109 57–64. Tiwary, P., Kumar, D., Mishra, M., Singh, R.P., Rai, M., Sundar, S., 2013. Seasonal variation in the prevalence of sand flies infected with Leishmania
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donovani 8, 1–8. doi:10.1371/journal.pone.0061370 Weirather, J.L., Jeronimo, S.M.B., Gautam, S., Sundar, S., Kang, M., Kurtz, M. a., Haque, R., Schriefer, A., Talhari, S., Carvalho, E.M., Donelson, J.E., Wilson, M.E., 2011. Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples. J. Clin. Microbiol. 49, 3892–3904. doi:10.1128/JCM.r00764-11 World Health Organization, 2010. Control of Leishmaniases: Report of a meeting of the WHO Expert Committee on the Control of Leishmaniases 22–26.
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Figure 1. A: Standard curve and linear regression of 10-fold dilutions of L. infantum DNA promastigotes mixed with 30 ng of DNA from reared sand flies. Mean Ct values and SDs of the three replicates of each dilution are shown. B: Graph of fluorescence results and number of cycles of each promastigote dilution (promastigote/ml) (1 promastigote/ml corresponds to 0.001 parasite/reaction), NTC (non template control) and NC (Negative control, DNA from reared sand fly).
Figure 2. qPCR amplification of samples. A: Representative fluorescence acquisition graph showed discrimination between negative and positive sand fly samples. NTC (non template control), NC (negative control corresponding to 30 ng of DNA from reared sand fly) and standards (106 and 103 promastigote/ml) were added (1 promastigote/ml corresponds to 0.001 parasites/reaction). B: Graphic showing concentration results of each sample according to standards. Slope = - 3.33; efficiency=0.99.
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Fig. 1
22
Fig. 2
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Table 1. Mean Ct values and SDs of standard curves of 10-fold dilutions series of promastigotes DNA and dilutions of promastigotes spiked with 30 ng of DNA from reared sand flies.
Mean Ct value ± SD
10-fold dilutions (Promastigotes/ml)
1
10
102
103
104
105
106
107
Promastigotes
25.99±0.29
22.57±0.15
22.27±0.21
20.81±0.16
17.92±0.3
14.4±0.13
10.91±0.31
7.30±0.15
Promastigotes + DNA from reared sand flies
25.23±0.57
22.24±0.34
21.53±0.27
20.29±0.28
17.57±0.35
14.03±0.46
10.68±0.24
7.24±0.29
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Table 2. Estimated parasite loads in blood-fed and unfed sand flies captured in each transmission period in the Leishmania outbreak of Madrid (Spain).
Estimated parasites/reaction >100,000 10,000>100,000 1,000>10,000 100>1,000 10>100 1>10 0.1>1 0.01>0.1 Total
Blood-fed sand flies 2012 1 0 3 5 2 3 7 2 23
2013 0 0 2 1 2 2 1 0 8
2014 0 0 0 0 1 2 1 0 4
Unfed sand flies 2012 0 6 4 3 1 0 0 0 14
2013 0 2 5 0 1 1 0 0 9
2014 0 2 2 3 2 0 1 0 10
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S0001-706X(16)31049-X http://dx.doi.org/doi:10.1016/j.actatropica.2017.03.023 ACTROP 4249
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
2-12-2016 20-3-2017 24-3-2017
´ Please cite this article as: Gonz´alez, Estela, Alvarez, Ana, Ruiz, Sonia, Molina, Ricardo, Jim´enez, Maribel, Detection of high Leishmania infantum loads in Phlebotomus perniciosus captured in the leishmaniasis focus of southwestern Madrid region (Spain) by real time PCR.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.03.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Detection of high Leishmania infantum loads in Phlebotomus perniciosus captured in the leishmaniasis focus of southwestern Madrid region (Spain) by real time PCR Estela González, Ana Álvarez, Sonia Ruiz, Ricardo Molina, Maribel Jiménez§ Unidad de Entomología Médica, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo s/n, 28220 Majadahonda, Madrid, Spain §
Corresponding author. Tel.: +34 91 822 3674; fax: +34 91 509 7034.
E-mail address: [email protected] (Maribel Jiménez)
Graphical abstarct In this work a qPCR assay was applied in order to estimate the number of parasites in P. perniciosus females collected in the human leishmaniasis focus that is affecting southwestern Madrid region
1
Highlights
A real time PCR was applied in female sand flies captured in a human leishmaniasis focus
Limit of detection achieved was 0.001 parasite/reaction, corresponding to 0.08fg of parasite DNA
High parasite loads (>1000 parasites/reaction) in sand flies from the focus were observed
Unfed females showed significant higher parasite loads than blood-fed females
Abstract Since 2010 a human leishmaniasis outbreak has been notified in southwestern Madrid region that still remains active. Entomological surveys have been carried out in the affected area in order to obtain information about species diversity, distribution, and density of sand flies. Moreover, molecular identification of blood meal preferences of sand flies and molecular detection of Leishmania infantum has been performed. In this work, we optimized a real time PCR assay in order to determine parasite loads in unfed and blood-fed Phlebotomus perniciosus female sand flies caught in the focus area. Results showed elevated parasite loads in nearly 70% of the studied positive sand flies. Furthermore, significantly higher parasite loads were observed in females without blood in their guts. In conclusion, high L. infantum loads found in P. perniciosus sand flies from the Madrid focus support the exceptional characteristics of this outbreak. 2
Keywords: Phlebotomus perniciosus, qPCR, Leishmania infantum loads, human leishmaniasis outbreak of Madrid
1. Introduction Leishmaniasis is a globally spread vector-borne disease caused by trypanosomatids of genus Leishmania and transmitted by phlebotomine sand fly bites. Southern Europe region is considered as a hypoendemic region for leishmaniasis (World Health Organization, 2010). In particular, Spain has registered an increase of visceral and cutaneous leishmaniasis human cases since 2010 when an outbreak of this disease located in the southwest of the Community of Madrid was detected (Arce et al., 2013; Gomez-Barroso et al., 2015). Since then, several studies have been performed in order to characterize this outbreak. Different factors related to the urbanization and changes in land use in the affected area of Madrid have been involved in the hypothesis about this leishmaniasis re-emergence, favoring the parasite transmission among wild animals of the green areas surrounding the municipalities affected by the outbreak. In this way, studies carried out elsewhere suggest that human intervention in many areas leads to a change in the transmission cycle of parasites by increasing exposure of infected vectors and susceptible reservoirs (Harhay et al., 2011). Hares and rabbits from the aforementioned region have been incriminated in the transmission of Leishmania infantum, demonstrating that a sylvatic cycle has been taking place (Jiménez et al., 2014; Molina et al., 2012). On the other hand, Leishmania parasites isolated from patients from the 3
focus, as well as from wild reservoirs and sand flies collected in the same area, have been characterized by molecular procedures showing the same genotype of L. infantum (Chicharro et al., 2013; Jiménez et al., 2014; Molina et al., 2012). Additionally, a great virulence of the L. infantum strain circulating in the focus area has been reported (Domínguez-Bernal et al., 2014). Systematic entomological captures have been carried out in the focus since 2011 demonstrating that the predominant sand fly species is Phlebotomus perniciosus (Alten et al., 2016; Tello et al., 2015), which is the main vector of leishmaniasis in Spain. Both molecular detections by conventional PCR and dissections performed with P. perniciosus females collected throughout these surveys show high L. infantum infection rates, although parasite loads have not been determined (Jiménez et al., 2014, 2013; data not published). At present, many studies have been performed for diagnostic purposes by using real time PCR (qPCR) assay for Leishmania detection and quantification. Thus, a qPCR targeting kDNA detection in fresh blood and tissue has also been developed by Mary et al., (2004) and Nicolas et al., (2002). Similarly, Abbasi et al., (2013) developed a qPCR in order to detect and quantify Leishmania donovani in human dried-blood. Leishmania species discrimination has been studied by qPCR in human samples (Weirather et al., 2011). Moreover, parasite loads in spleen have been correlated with blood parasite burden in visceral leishmaniasis using qPCR (Sudarshan et al., 2015). Even more, some authors searching for non-invasive methods have developed qPCR assays in order to detect and quantify Leishmania parasites in hair and earwax of dogs (Belinchón-Lorenzo et al., 2016, 2013). On the other hand, there are several works focused on the study of parasite burden in sand fly vectors. Myskova et
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al., (2008) describe for the first time a qPCR method to determine the parasite loads in Phlebotomus duboscqi. Since then, qPCR has been applied in other sand fly species such as Phlebotomus sergenti of Iran (Aghaei et al., 2014); Phlebotomus papatasi and Phlebotomus alexandri of Iraq (Coleman et al., 2009), Phlebotomus perfiliewi, P. perniciosus, and Phlebotomus neglectus of Italy (Dantas-Torres et al., 2014), and Lutzomyia longipalpis of Brazil (BezerraVasconcelos et al., 2011; Cunha et al., 2014). Undoubtedly, knowledge of parasite loads in sand flies may provide important information about transmission patterns in a particular region. Furthermore, it is well known that Leishmania parasites can manipulate sand fly feeding behavior. Thus, high parasite loads in sand fly midguts are correlated to a persistent feeding pattern that leads to an increase in Leishmania transmission (Rogers and Bates, 2007). In the present work, we applied and validated a qPCR assay to detect and estimate the number of L. infantum parasites in the gut of P. perniciosus females caught in the aforementioned human leishmaniasis focus that is affecting southwestern Madrid region.
2. Materials and Methods 2.1.
Leishmania parasites
The L. infantum strain IPER/ES/2012/POL2FL7, isolated from a naturally infected sand fly captured in the leishmaniasis focus of southwestern Madrid region (Spain) and characterized as ITS-Lombardi genotype, was used in this study. The promastigotes were cultured in RPMI medium supplemented with 20 % inactivated fetal calf serum (FCS, HycloneTM, GE Healthcare Life Science, NJ, USA) and a mixture of penicillin-streptomycin (10000 U/ml, Lonza 5
BioWitthaker®, Verviers, Belgium). L. infantum promastigotes were collected by centrifugation and resuspended in buffered saline. Then, promastigotes suspension was quantified by duplicate using a Neubauer® chamber in order to obtain 107 promastigotes/ml. Subsequent 10-fold dilutions were carried out. 2.2.
Samples
DNA samples obtained from 135 female sand flies collected in the entomological surveys carried out from June to October 2012, 2013, and 2014 were analyzed by qPCR. These samples were previously examined by conventional kDNA-PCR finding 68 positive sand flies for Leishmania DNA. Sixty-four out of the 135 analyzed sand flies showed indication of a recent blood meal. Sand flies reared in the Medical Entomology Unit of the Carlos III Health Institute, Spain, were used as negative control. 2.3.
DNA isolation
DNA from promastigotes dilutions were extracted using Blood and Tissue DNA extraction Kit (Qiagen®) following manufacturer instructions. On the other hand, DNA from reared and field-captured sand flies was obtained using the abovementioned kit and following the protocol previously described (Jiménez et al., 2013). 2.4.
Determination of parasite loads by qPCR
Kinetoplast minicircle primers JW11: 5’-CCT ATT TTA CAC CAA CCC CCA GT-3’ and JW12: 5’-GGG TAG GGG CGT TCT GCG AAA-3’ (Nicolas et al., 2002) were used for Leishmania detection in both conventional and qPCR methods. Different assays were performed in order to enhance primer
6
concentration and the number of cycles of qPCR. Finally, each reaction consisted in a final volume of 20 µl, containing 10 µl of Quantimix Easy Kit (Biotools®), 0.1 µM of each primer, 0.5 µl of BSA (Roche®), and 30 ng of DNA extracted from each sand fly. PCR amplification was performed as follows: an initial incubation at 95ºC for 15 min followed by 40 cycles at 95ºC for 10 s, 58ºC for 10 s and a final extension at 72ºC for 30 s. At the end of each cycle, SYBR® Green I fluorescence acquisition was performed. Non template control (NTC), negative control (NC, reared sand flies) and positive controls (two promastigotes dilutions mixed with 30 ng of sand fly DNA) were added in each reaction. Amplifications were carried out in a Corbett Rotor-GeneTM 6000 realtime PCR System (Qiagen®). 2.5.
Data analysis and statistics
Threshold cycle (Ct) values were calculated by Rotor-Gene Series Software version 1.7 using default parameters. Standard curves using DNA obtained from promastigotes dilutions and DNA from promastigotes dilutions mixed with 30 ng of DNA from reared sand flies were performed by triplicate. Mean and standard deviation (SD) of Ct values of each standard curve dilution were calculated. Non parametric statistical Kruskal-Wallis method was used to calculate significant differences between Ct values in the three replicates of each dilution of the standard curves. The same statistical test was applied to study differences in parasite loads between the three periods. Meanwhile, MannWhitney method was used to study differences between Ct values of both standard curves. This statistical method was also applied to analyze parasite loads differences among blood-fed and unfed female sand flies. Differences 7
were considered statistically significant when p-values were less than 0.05, using GraphPad Prism 6.07 software.
3. Results 3.1.
Sensitivity and Efficiency
With the aim to optimize this qPCR, several experiments were carried out using different primer concentration and number of cycles per reaction. In this assay, we observed that a number of cycles greater than 40 produced a visible smear in the agarose gel and a lower efficiency. On the other hand, a primer concentration higher than 0.1 µM resulted in dimers presence. To analyze the sensitivity of the reaction, amplification of 10-fold dilutions ranged from 107 to 1 promastigote/ml was carried out by triplicate. The limit of detection (LOD) was 0.001 parasite/reaction, which corresponds to 1 promastigote/ml from the original sample (1 µl was used per reaction). A second standard curve mixing DNA extracted from each promastigote dilution and 30 ng of DNA from reared sand flies was performed. The LOD obtained in both standard curves was the same. Based on the haploid genome size of L. infantum (32.1Mb, 69.7 fg) and adding ≃10.5 fg of kinetoplast genomic material, one parasite yield ≃80.2 fg (Peacock et al., 2007). According to this, our qPCR assay achieved a LOD of 0.08 fg of parasite DNA. On the other side, reproducibility in both standard curves was assessed and replicates of each dilution did not show significant differences according to statistical results (pvalues = 0.8981 and 0.8847 for DNA promastigote dilutions and DNA from promastigotes mixed with sand fly DNA, respectively). In the same way, there 8
was no significant difference between both standard curves (p-value = 0.6294), showing no inhibition of DNA from reared sand flies in the reaction (Table 1). Finally, standard curves showed loss of linearity and efficiency when dilutions below 1 parasite/reaction were used (Fig. 1). The efficiency showed optimum values (average slope= -3.2±0.14; average efficiency= 1.06±0.07) in case of qPCR reactions of samples (Fig. 2). 3.2.
Leishmania quantification in field-captured sand flies
A total of 135 DNA samples of female sand flies captured in the outbreak area in southwestern Madrid were analyzed by the qPCR optimized in this study. Results by qPCR did not present differences with conventional PCR, detecting n= 68 positive samples (35 blood-fed and 33 unfed) and n= 67 negative samples (29 blood-fed and 38 unfed females) by both methods. Ct threshold values were calculated according to default parameters and NTC Ct values in each qPCR reaction were employed as the cut-off value for discrimination between positive and negative samples (Fig. 2). The 68 positive samples were classified in different categories according to the parasite loads: 16.17% (n=11) showed very high loads (>10,000 parasites/reaction; a specimen showing over than 100,000 parasites/reaction is included in this category), 23.53% (n = 16) showed high loads (>1,000 parasites/reaction), 30.9% (n = 21) presented moderate loads (10-1000 parasites/reaction) and 29.4% (n = 20), low parasite loads (<10 parasites/reaction) (Table 2). Parasite burden were higher in sand flies caught in 2012, although differences were not significant (p-value = 0.7233).
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Mean parasite load in blood-fed females was 8.02 parasites/reaction, while in unfed females was 1,580 parasites/reaction. In this case, the differences are significant (p-value < 0.0001).
4. Discussion This study presents the validation of a qPCR assay for the detection of L. infantum and the quantification of parasite loads using sand flies collected in a periurban area of southwestern Madrid affected by a leishmaniasis outbreak since 2010 (Arce et al., 2013; Gomez-Barroso et al., 2015). A high abundance of P. perniciosus sand fly, the most important vector of leishmaniasis in western Mediterranean basin, has been found in this focus (Aránguez et al., 2014; Arce et al., 2013; Tello et al., 2015; Alten et al., 2016). Additionally, other studies revealed some special features of this focus like the existence of a sylvatic cycle of L. infantum involving lagomorphs living in the green areas surrounded by the aforementioned urban area (Jiménez et al., 2013, 2014; Martín-Martín et al., 2014; Molina et al., 2012) and the high virulence of the L. infantum isolates obtained from sand flies captured in the same areas (Domínguez-Bernal et al., 2014; Martín-Martín et al., 2015). Even though elevated Leishmania infection rates have been observed by dissection (data not published) and conventional PCR (Jiménez et al., 2013) among P. perniciosus females collected in the focus, no information was available on their parasite loads to date. In the present work, the qPCR showed the same sensitivity as the conventional PCR performed on the same samples. The LOD has been established in 0.001 parasites/reaction with the qPCR developed, corresponding to 0.08 fg of parasite DNA. Similar LOD values have been reported by others using qPCR in 10
sand flies targeting kDNA (Bezerra-Vasconcelos et al., 2011; Cunha et al., 2014; Myskova et al., 2008). In spite of the good LOD, standard curves showed less linearity in dilutions lower than 1 parasite/µl, as has been reported by Abbasi et al., (2013) when assessing the efficacy of qPCR for detecting L. donovani in human dried-blood samples. Nevertheless, reactions performed in the present work with sand fly samples demonstrated good efficiency and parasite burden was successfully estimated. Seventy percent of the sand flies analyzed in this study presented a significant number of Leishmania parasites in their guts and 16.17% of them showed large infections greater than 10,000 parasites/reaction, and what is more, a specimen presented more than 100,000 parasites/reaction. Such parasite loads found in wild caught P. perniciosus females had not been reported so far. Only studies carried out in wild L. longipalpis have reported higher parasite loads (Rodrigues et al., 2016). The exceptional features observed in the outbreak area as the high density of lagomorphs (Jiménez et al., 2014), the high L. infantum infection rates in spleen and skin samples of hares (29.9%) and rabbits (21.2%) (Community of Madrid data), and the elevated proportion of sand flies engorged with blood of rabbits or hares in the green park (Jiménez et al., 2013, 2014; González et al., 2015), may have given rise to the high parasite loads detected in wild caught sand flies, thereby increasing the transmission rates of the parasite. In addition, a large number of promastigotes can block more easily the stomodeal valve of sand flies forcing them to remain feeding for longer on the same or different hosts to often obtain only a partial meal of blood which increases the likelihood of parasite transmission. In this sense, experimental infections have proven that both L. infantum and L. mexicana promote feeding 11
on multiple hosts (Rogers and Bates, 2007). Moreover, the infectious dose may be one of the determining factors in the outcome of Leishmania infection (Kimblin et al., 2008; Maia et al., 2011). The significantly higher parasite loads found in females without blood in their guts are undeniable proof of a proper establishment and replication of the parasites that would be more likely to survive and develop late-stage infections in the sand fly midgut (Roger and Bates, 2007, Myskova et al., 2008). The intensity of infection in wild caught sand flies gives evidence of parasite growth and possible transmission (Killick-Kendrick, 1990). In other words, P. perniciosus is an excellent vector of leishmaniasis in the outbreak area. Similarly, other authors have found in India higher parasite loads in gravid and unfed sand flies than in blood-fed sand flies (Tiwary et al., 2013). As for the low parasite loads found in most of the blood-engorged sand flies studied could be due to a recent ingestion of blood on infecting vertebrates from which these uninfected flies had fed. Finally, the high parasite loads observed in some females with freshly ingested blood in their guts suggest that they could be already infected at the time of the next blood ingestion and potentially transmit the parasite to the vertebrate from which they had fed. These findings could give us an idea about the intensity of transmission of the parasite from wild vertebrates to sand flies in the area. Although the quantification of parasite burden in sand flies by qPCR can be suitable for the incrimination of suspected vectors of leishmaniasis, it would be advisable to combine with sand fly dissection in order to determine the localization of the promastigotes in the gut and to isolate the leishmania strain (Myskova et al., 2008).
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In conclusion, the qPCR assay validated in the present study has allowed the detection and quantification of elevated parasite loads in a high number of the P. perniciosus female sand flies captured in the human leishmaniasis focus that is affecting southwestern Madrid region. These results are further evidence of the exceptional features of this leishmaniasis focus which require special attention by establishing an intensive and ongoing surveillance program of the disease in the outbreak area.
Conflict of interest The authors declare no conflicts of interest.
Acknowledgments This study was partially funded by the Dirección General de Salud Pública, Consejería de Sanidad of Community of Madrid, Spain and the Instituto de Salud Carlos III, Madrid, Spain. We want to thank Sonia Hernández for technical assistance.
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Figure 1. A: Standard curve and linear regression of 10-fold dilutions of L. infantum DNA promastigotes mixed with 30 ng of DNA from reared sand flies. Mean Ct values and SDs of the three replicates of each dilution are shown. B: Graph of fluorescence results and number of cycles of each promastigote dilution (promastigote/ml) (1 promastigote/ml corresponds to 0.001 parasite/reaction), NTC (non template control) and NC (Negative control, DNA from reared sand fly).
Figure 2. qPCR amplification of samples. A: Representative fluorescence acquisition graph showed discrimination between negative and positive sand fly samples. NTC (non template control), NC (negative control corresponding to 30 ng of DNA from reared sand fly) and standards (106 and 103 promastigote/ml) were added (1 promastigote/ml corresponds to 0.001 parasites/reaction). B: Graphic showing concentration results of each sample according to standards. Slope = - 3.33; efficiency=0.99.
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Fig. 1
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Fig. 2
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Table 1. Mean Ct values and SDs of standard curves of 10-fold dilutions series of promastigotes DNA and dilutions of promastigotes spiked with 30 ng of DNA from reared sand flies.
Mean Ct value ± SD
10-fold dilutions (Promastigotes/ml)
1
10
102
103
104
105
106
107
Promastigotes
25.99±0.29
22.57±0.15
22.27±0.21
20.81±0.16
17.92±0.3
14.4±0.13
10.91±0.31
7.30±0.15
Promastigotes + DNA from reared sand flies
25.23±0.57
22.24±0.34
21.53±0.27
20.29±0.28
17.57±0.35
14.03±0.46
10.68±0.24
7.24±0.29
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Table 2. Estimated parasite loads in blood-fed and unfed sand flies captured in each transmission period in the Leishmania outbreak of Madrid (Spain).
Estimated parasites/reaction >100,000 10,000>100,000 1,000>10,000 100>1,000 10>100 1>10 0.1>1 0.01>0.1 Total
Blood-fed sand flies 2012 1 0 3 5 2 3 7 2 23
2013 0 0 2 1 2 2 1 0 8
2014 0 0 0 0 1 2 1 0 4
Unfed sand flies 2012 0 6 4 3 1 0 0 0 14
2013 0 2 5 0 1 1 0 0 9
2014 0 2 2 3 2 0 1 0 10
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